Methods of using farnesoid x receptor (frx) agonists

ABSTRACT

Treatment of human hepatocytes with farnesoid X receptor (FXR) agonists resulted in increased expression of FGF-19. Methods of using FXR agonists to alter cell metabolism, and in pharmaceutical weight loss methods, are described.

FIELD OF THE INVENTION

The present invention relates to farnesoid X receptor (FXR) agonists andtheir use in methods of affecting the metabolism of cells, and inpharmaceutical weight loss methods.

BACKGROUND OF THE INVENTION

Being overweight or obese substantially raises an individual's risk ofmorbidity from hypertension, dyslipidemia, type 2 diabetes, coronaryheart disease, and other conditions. Despite the expected medicalbenefits, many overweight individuals find it difficult to successfullylose weight by diet management alone. Obesity is recognized as a complexmultifactorial condition that develops from the interaction of geneticand environmental factors. See, e.g., Clinical Guidelines on theIdentification, Evaluation, and Treatment of Overweight and Obesity inAdults, Am. J. Clin. Nutr. 68:899 (1998).

Various pharmaceutical compounds have been utilized in weight losstreatments. Serotonergic agents that inhibit the reuptake of serotoninare reported to act on the hypothalamus to decrease satiety. However,serious cardiovascular side effects have been reported in someindividuals treated with such agents. Fenfluramine and dexfenfluramine,serotonergic agents previously utilized in the United States for thetreatment of obesity, have been withdrawn from the U.S. market due toreports of valvular heart disease and primary pulmonary hypertension.(Davidoff et al., Arch Intern Med 161:1429 (2001); Michelakis et al., AmJ Med Sci 321:292 (2001); Weissman, Am J Med Sci 321:285 (2001); 2001PHYSICIANS DESK REFERENCE®, Medical Economics Co., (2000)).

In view of the need for medical weight loss therapies, additionalpharmaceutical methods useful in weight control or weight loss aredesirable.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method of increasing leptinrelease from the adipocyte cells of a mammalian subject, byadministering an FXR agonist to the subject. Leptin release isincreased, compared to the leptin release that would occur without FXRagonist administration.

A further aspect of the present invention is a method of decreasingglucose uptake by the adipocyte cells of a mammalian subject, byadministering an FXR agonist to the subject. Glucose uptake is decreasedcompared to that which would occur in the absence of FXR agonistadministration.

A further aspect of the present invention is a method of treating amammalian subject to achieve weight loss, by administration of apharmaceutically acceptable FXR agonist. The subject's weight isdecreased, compared to that which would occur in the absence of FXRagonist treatment.

A further aspect of the present invention is a method of reducing thetotal body mass of a mammalian subject, by administering apharmaceutically acceptable FXR agonist. The subject's total body massis reduced compared to the subject's total body mass that would occur inthe absence of FXR agonist treatment.

A further aspect of the present invention is a method of increasing themetabolic rate of a mammalian subject, by administering apharmaceutically acceptable FXR agonist. The metabolic rate of thesubject is increased compared to the rate that would occur in theabsence of FXR agonist treatment.

A further aspect of the present invention is a method of increasingserum leptin in a mammalian subject, by administering to a subject apharmaceutically acceptable FXR agonist. The serum leptin in the subjectis thereby increased compared to that would occur in the absence of FXRagonist treatment.

A further aspect of the present invention is a method of inducingexpression of FGF19 in a human hepatocyte cell, by administering an FXRagonist to the cell. The cell maybe in vitro.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of Farnesoid X Receptor (FXR)agonists to affect the metabolism of cells, and as a pharmaceuticaltreatment for weight control and weight loss. The present inventorsdetermined that activation of the nuclear receptor FXR by a bile acidagonist, as well as by a small molecule FXR agonist, caused an increasein transcription of a human Fibroblast Growth Factor gene (hFGF19),leading to an increase in the quantity of mRNA encoding the fibroblastgrowth factor. Accordingly, in humans expression of FGF19 and itsdownstream activity can be modulated using FXR agonists. Human FGF19 hasbeen reported to induce leptin release from rat adipocyte cells anddecrease glucose uptake by rat adipocyte cells; transgenic miceexpressing hFGF19 have been reported to be less fat than theirnontransgenic littermates; increased oxygen consumption has beenreported in mice administered recombinant FGF-19; and administration ofFGF-19 has been suggested as a treatment for obesity (WO 01/18210,Genentech).

FXR

FXR is a member of the nuclear receptor family of ligand-activatedtranscription factors that includes receptors for the steroid, retinoid,and thyroid hormones (D J. Mangelsdorf, et al., Cell 83:841-850 (1995)).Northern and in situ analysis show that FXR is most abundantly expressedin the liver, intestine, kidney, and adrenal (B M. Forman, et al., Cell81:687 (1995) and W. Seol, et al., Mol. Endocrinnol. 9:72 (1995)). FXRbinds to DNA as a heterodimer with the 9-cis retinoic acid receptor(RXR). The FXR/RXR heterodimer preferentially binds to response elementscomposed of two nuclear receptor half sites of the consensus AG(G/T)TCAorganized as an inverted repeat and separated by a single nucleotide(IR-1 motif) (B M. Forman, et al., Cell 81:687 (1995)). An early reportshowed that rat FXR is activated by micromolar concentrations offarnesoids such as farnesol and juvenile hormone (B M. Forman, et al.,Cell 81:687-693 (1995)). However, these compounds failed to activate themouse and human FXR, leaving the nature of the endogenous FXR ligand indoubt. Several naturally-occurring bile acids bind to and activate FXRat physiological concentrations (PCT WO 00/37077, published 29 Jun.2000)). As discussed therein, the bile acids that serve as FXR ligandsinclude chenodeoxycholic acid (CDCA), deoxycholic acid (DCA),lithocholic acid (LCA), and the taurine and glycine conjugates of thesebile acids.

Bile acids are cholesterol metabolites that are formed in the liver andsecreted into the duodenum of the intestine, where they have roles inthe solubilization and absorption of dietary lipids and vitamins. Mostbile acids (˜95%) are subsequently reabsorbed in the ileum and returnedto the liver via the enterohepatic circulatory system. The conversion ofcholesterol to bile acids in the liver is under feedback regulation:bile acids down-regulate the transcription of cytochrome P450 7a(CYP7a), which encodes the enzyme that catalyzes the rate limiting stepin bile acid biosynthesis. FXR is involved in both the stimulation andthe repression (via CYP7a) of target genes involved in bile acid andcholesterol homeostasis.

FXR Ligands

The bile acids chenodeoxycholic acid (CDCA), deoxycholic acid (DCA),lithocholic acid (LCA), and the taurine and glycine conjugates thereofselectively activate FXR (WO 0037077, Glaxo Group Limited). As usedherein, the term “FXR agonist” refers to compounds that achieve at leastabout 50% activation of FXR relative to CDCA, the appropriate positivecontrol in the assay methods described in PCT Publication No. WO00/37077 published 29 Jun. 2000 to Glaxo Group Limited, the subjectmatter of which is incorporated herein by reference in its entirety.Preferably, the FXR agonist compounds used in the methods of thisinvention achieve at least about 70% activation of FXR in thescintillation proximity assay or the HTRF assay as described in PCTPublication No. WO 00/37077; more preferably, the compounds achieve atleast about 80%, 90%, 95%, 97% or greater activation of FXR in thescintillation proximity assay or the HTRF assay as described in PCTPublication No. WO 00/37077.

An FXR agonist for use in the present invention is the compound known asGW4064, as disclosed in PCT Publication No. WO 00/37077 published 29Jun. 2000 to Glaxo Group Limited, which describes FXR ligand compoundscharacterized by the following formula (I)

wherein X¹ is CH or N; X² is O or NH; R and R¹ may independently be H,lower alkyl, halogen, or CF₃; R² is lower alkyl; R³ and R⁴ mayindependently be H, lower alkyl, halogen, CF₃, OH, O-alkyl, orO-polyhaloalkyl.

GW4064, an example of a compound of Formula (I), is a potent andselective FXR ligand and has the following formula (II):

FXR agonists for use in the present invention further include compoundsof formula III:

wherein R is ethyl, propyl or allyl, and pharmaceutically acceptablesalts, solvates or amino acid conjugates thereof.

Suitable pharmaceutically acceptable salts of the above compounds may bereadily determined by one skilled in the art and may include, forexample, basic salts such as metallic salts made from aluminium,calcium, lithium, magnesium, potassium, sodium, and zinc or organicsalts made from N,N′-dibenzylethylenediamine, chlorprocaine, choline,diethanolamine, ethylendiamine, meglumine (N-methylglucamine), andprocaine. Such salts may be prepared using conventional techniques, asare known in the art. As used herein, the tern “solvate” is a crystalform containing the active compound (or a pharmaceutically acceptablesalt thereof) and either a stoichiometric or a non-stoichiometric amountof a solvent. Solvents, by way of example, include water, methanol,ethanol, or acetic acid.

As used herein, the term “amino acid conjugates” refers to conjugates ofa compound with any suitable amino acid. Preferably, such suitable aminoacid conjugates have the added advantage of enhanced integrity in bileor intestinal fluids. Suitable amino acids include but are not limitedto glycine and taurine. Thus, the present invention encompasses the useof glycine and taurine conjugates of FXR agonists.

Compounds of formula (III) include compounds selected from the groupconsisting of 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid;3α,7α-dihydroxy-6α-propyl-5β-cholan-24-oic acid and3α,7α-dihydroxy-6α-allyl-5β-cholan-24-oic acid and theirpharmaceutically acceptable salts, solvates or amino acid conjugatesthereof.

The amount of FXR agonist, or pharmaceutically acceptable salt orsolvate thereof, which is required to achieve the desired biologicaleffect will depend on a number of factors such as the means ofadministration, the desired outcome, and the recipient. In general, intreating mammals for weight control or weight loss purposes, a typicaldaily dose of an FXR agonist of formula (I-III) may be expected to liein the range of from about 0.01 mg/kg to about 100 mg/kg. This dose maybe administered as a single unit dose or as several separate unit dosesor as a continuous infusion.

FGF19

FGF19 is a member of the fibroblast growth factor (FGF) family ofproteins. Members of this family are known to be involved in tissuerepair, angiogenesis, induction of genes containing an FGF-inducibleresponse element (FiRE), mitogenesis, oncogenesis, and differentiation.The biological specificity of FGFs is believed to be partly due to thecontrolled expression of both the FGFs and the FGF receptors (FGFRs).Four related receptor tyrosine kinases have been identified that bind tomembers of the FGF family; the presence of heparin or heparan sulfateproteoglycans is believed to be required for the biological activity ofFGFs.

The structure and expression of human FGF-19 is described in Nishimuraet al., Biochim Biophys Acta 1444:148 (1999). FGF-19 binds with highaffinity to the cell surface tyrosine kinase receptor FGF Receptor 4(FGFR4), and displays selective binding to FGFR4. Xie et al., Cytokine11:729 (1999).

The sequence for human FGF19 mRNA is provided at GenBank AccessionNumber AF110400: CDS 464-1114 gctcccagcc aagaacctcg gggccgctgcgcggtgggga ggagttcccc gaaacccggc 60 (SEQ ID NO:1) cgctaagcga ggcctcctcctcccgcagat ccgaacggcc tgggcggggt caccccggct 120 gggacaagaa gccgccgcctgcctgcccgg gcccggggag ggggctgggg ctggggccgg 180 aggcggggtg tgagtgggtgtgtgcggggg gcggaggctt gatgcaatcc cgataagaaa 240 tgctcgggtg tcttgggcacctacccgtgg ggcccgtaag gcgctactat ataaggctgc 300 cggcccggag ccgccgcgccgtcagagcag gagcgctgcg tccaggatct agggccacga 360 ccatcccaac ccggcactcacagccccgca gcgcatcccg gtcgccgccc agcctcccgc 420 acccccatcg ccggagctgcgccgagagcc ccagggaggt gccatgcgga gcgggtgtgt 480 ggtggtccac gtatggatcctggccggcct ctggctggcc gtggccgggc gccccctcgc 540 cttctcggac gcggggccccacgtgcacta cggctggggc gaccccatcc gcctgcggca 600 cctgtacacc tccggcccccacgggctctc cagctgcttc ctgcgcatcc gtgccgacgg 660 cgtcgtggac tgcgcgcggggccagagcgc gcacagtttg ctggagatca aggcagtcgc 720 tctgcggacc gtggccatcaagggcgtgca cagcgtgcgg tacctctgca tgggcgccga 780 cggcaagatg caggggctgcttcagtactc ggaggaagac tgtgctttcg aggaggagat 840 ccgcccagat ggctacaatgtgtaccgatc cgagaagcac cgcctcccgg tctccctgag 900 cagtgccaaa cagcggcagctgtacaagaa cagaggcttt cttccactct ctcatttcct 960 gcccatgctg cccatggtcccagaggagcc tgaggacctc aggggccact tggaatctga 1020 catgttctct tcgcccctggagaccgacag catggaccca tttgggcttg tcaccggact 1080 ggaggccgtg aggagtcccagctttgagaa gtaactgaga ccatgcccgg gcctcttcac 1140 tgctgccagg ggctgtggtacctgcagcgt gggggacgtg cttctacaag aacagtcctg 1200 agtccacgtt ctgtttagctttaggaagaa acatctagaa gttgtacata ttcagagttt 1260 tccattggca gtgccagtttctagccaata gacttgtctg atcataacat tgtaagcctg 1320 tagcttgccc agctgctgcctgggccccca ttctgctccc tcgaggttgc tggacaagct 1380 gctgcactgt ctcagttctgcttgaatacc tccatcgatg gggaactcac ttcctttgga 1440 aaaattctta tgtcaagctgaaattctcta attttttctc atcacttccc caggagcagc 1500 cagaagacag gcagtagttttaatttcagg aacaggtgat ccactctgta aaacagcagg 1560 taaatttcac tcaaccccatgtgggaattg atctatatct ctacttccag ggaccatttg 1620 cccttcccaa atccctccaggccagaactg actggagcag gcatggccca ccaggcttca 1680 ggagtagggg aagcctggagccccactcca gccctgggac aacttgagaa ttccccctga 1740 ggccagttct gtcatggatgctgtcctgag aataacttgc tgtcccggtg tcacctgctt 1800 ccatctccca gcccaccagccctctgccca cctcacatgc ctccccatgg attggggcct 1860 cccaggcccc ccaccttatgtcaacctgca cttcttgttc aaaaatcagg aaaagaaaag 1920 atttgaagac cccaagtcttgtcaataact tgctgtgtgg aagcagcggg ggaagaccta 1980 gaaccctttc cccagcacttggttttccaa catgatattt atgagtaatt tattttgata 2040 tgtacatctc ttattttcttacattattta tgcccccaaa ttatatttat gtatgtaagt 2100 gaggtttgtt ttgtatattaaaatggagtt tgtttgtaaa aaaaaaaaaa aaaaaaa 2157

Human FGF19 polypeptides and nucleic acid molecules encoding the sameare described in WO 0118210 (Genentech, Inc.), EP1032668 (Genentech),and WO 0118209 (Curagen Corporation). The production of transgenic miceexpressing human FGF19 (using the promoter for myosin light chain toresult in muscle specific transcription of the transgene) is reported inWO 0118210, where it is further reported that these mice demonstratedincreased food intake and increased metabolic rate (evidenced by theirrate of oxygen consumption and increased urine output). However, thetransgenic mice weighed significantly less than their non-transgeniclittermates, despite their increased food intake. The transgenic micehad normal linear growth, and normal body temperature and bone length.It is postulated in WO0118210 that the decreased body weight oftransgenic hFGF19 mice is due to decreased adiposity; leptin (which isreported to correlate closely with adipose tissue mass in humans androdents) is decreased in the transgenic mice. Infusion of FGF19 tonon-transgenic mice was reported to cause an increase in food intake. Itis stated in WO0118210 that FGF19 decreases adiposity without alteringmuscle mass or long bone formation, and that FGF19 is indicated as atherapeutic in the treatment of obesity and related conditions. Further,the effects of a high-fat diet on glucose tolerance in transgenic miceexpressing hFGF19 were compared to the effects in non-transgeniclittermates. Transgenic rice fed a high-fat diet for ten weeks weresubjected to a glucose tolerance test; the majority of thenon-transgenic mice fed a high-fat diet were defined as diabetic,whereas none of the transgenic mice fed a comparable high-fat diet weredefined as diabetic by the glucose tolerance test. WO 01/18210.

Definitions

“Mammal” as used herein includes primates and humans, as well aslivestock and companion animals.

“Pharmaceutical weight loss treatment” as used herein refers toadministration of a pharmaceutical compound to a subject whose weight isgreater than a medically acceptable or medically desirable amount, toachieve a reduction in the subject's weight. “pharmaceutical treatmentof obesity” is an aspect of pharmaceutical weight loss treatment andrefers to such treatment for individuals whose body mass meets anaccepted medical definition of obesity. One commonly accepted measure ofoverweight in humans is the Body Mass Index (BMI); overweight may bedefined as a BMI of at least 25 kg/m², with obesity defined as a BMI ofat least 30 kg/m². Pharmaceutical weight loss treatment may beaccompanied by a change in diet and/or other behavioral modificationssuch as support groups and/or patient education. As used herein,pharmaceutical weight loss treatment does not imply a “cure” for obesityor permanent weight loss. “Pharmaceutical weight maintenance treatment”as used herein refers to administration of a pharmaceutical compound toa subject as an aid in maintaining a desired weight. As describedherein, FXR agonists are useful in such treatments described above.

Body Mass Index is a numerical measurement of relative weight forheight, and has been significantly correlated with total body fatcontent. BMI is calculated as weight (kg)/height squared (m²). See,e.g., Clinical Guidelines on the Identification, Evaluation, andTreatment of Overweight and Obesity in Adults. Am. J. Clin. Nutr. 68:899(1998).

As used herein, an FXR ligand pharmaceutical compound for the treatmentof obesity or for weight loss treatment is one in which administration(in an appropriate pharmaceutical formulation and in a therapeuticallyeffective amount) to a mammal, and preferably to humans, has been shownto increase weight loss over time, compared to the change in weight thatwould have occurred had the subject not been administered the compound.Therapeutically effective amounts of such compounds for use in treatmentof obesity or for weight loss can be readily determined by those skilledin the art using, e.g., dose-response studies.

Treatment of a subject with an FXR agonist pharmaceutical compoundcomprises administration of an effective amount (for the condition beingtreated) of the pharmaceutical agent to a subject. The dose of agent isdetermined according to methods known and accepted in the pharmaceuticalarts, and can be determined by those skilled in the art.

Differential Gene Expression

The present study utilized differential gene expression analysis asperformed by CuraGen Corporation (New Haven, Conn.) using transcriptprofiling technology. See Nat Biotechnol 17:798-803, 1999; see also U.S.Pat. Nos. 5,871,697 and 5,972,693. In brief, RNA is extracted fromsamples and cDNA synthesis is carried out. The cDNAs are then digestedwith multiple pairs of different restriction enzymes (standard set of 96different pairs of restriction enzymes with 6 base-pair recognitionsites (subsequence pairs)), and the digested products are ligated tocomplementary adapters containing standardized PCR priming sequences.Multiple PCR cycles are carried out using one biotinylatedadapter-specific primer and one fluorescently labeled adapter-specificprimer. Following PCR amplification, the biotin-labeled DNA is purifiedon immobilized streptavidin. Denatured single stranded cDNA fragmentsare then sized using capillary electrophoresis, and the fluorescentlylabeled fragments are detected by laser excitation. Since the biotinlabel is needed for purification and the fluorescent label is needed fordetection, all analyzed fragments result from restriction digestion withboth enzymes.

Differentially expressed peaks are identified by comparing the compositetraces of the experimental samples vs. the control samples usingbioinformatics algorithms. Each differentially expressed peak is thencompared to a database containing a ‘virtual’ restriction digest of ahuman sequence database. (The database was constructed by performing insilico digests on a human sequence database, using all subsequence pairsas described above; for each transcribed DNA sequence, the computedfragment sizes and associated restriction enzymes (bands) are stored inthe database.) By comparing the differentially expressed peaks with thedatabase, genes are identified that are predicted to generate DNAfragments with lengths and terminal sequences that match the restrictionenzymes used and the detected length of the differentially expressedpeaks. A single differentially expressed band may be part of one or moregenes. Additional bands from at least one of these genes should bepresent and should also be differentially expressed.

Thus, confirmation and gene identification of differentially expressedbands can occur by two routes. One method is GeneCalling/Poisoning. Inthis case, cDNA fragments representing differentially expressed genescan be identified by database searching with the 6 base-pair restrictionenzyme recognition sequences at the fragment ends and the exact lengthof each fragment (determined electrophoretically, subtracting linkerlength). Database searching for genes predicted to have restrictionfragments of matching lengths enables the identification of such geneswhose sequences reside in that database (a “GeneCall”). The detection ofmultiple fragments derived from the same gene which show differentialexpression of the same directional modulation increases the likelihoodthat the prediction of the gene identity is correct. The differentiallyexpressed gene fragment and the predicted gene sequence identified bythe database lookup, can then be linked through a positive poisoningreaction. In this process, the reaction containing the fragment ofinterest is performed a second time using the same end primers, but inthe presence or absence of an excess of an unlabeled oligonucleotidewhose sequence is derived from the predicted gene fragment. If theidentity of the fragment was predicted correctly, the unlabeledoligonucleotide will out-compete the universal oligonucleotide forpriming that fragment and, in the resulting chromatogram, will appear toablate that peak specifically without affecting the amplification of theother fragments.

An alternative method for gene identification and confirmation,Isolation/Poisoning, relies on the isolation of the differentiallyexpressed fragment from the re-amplified GeneCalling chemistry reactionfrom a preparative gel. The gel-purified fragment is re-amplified andcloned in a standard PCR product cloning vector. The insert is sized andsequenced, and primers for poisoning are designed from one or both endsof the cloned fragment. The poisoning reaction is performed and analyzedas described above. Successful ablation of the peak using the unlabeledoligonucleotide based on the cloned sequence identifies the sequence ascorresponding to the original differentially expressed gene fragment.Subsequently, the gene identification is obtained by standard BLASTN orBLASTX analysis of the poisoning cloned sequence.

The present invention provides methods of using FXR agonists to modulateFGF-19 expression by mammalian cells and, in mammalian organisms, tothereby modulate the downstream effects of FGF-19. The present methodsfurther provide methods of using FXR agonists to modulate the metabolismof mammals , and as a treatment for weight loss or weight control inmammals. Such methods include using FXR agonist administration to amammal to achieve an increase in circulating or serum leptin levels. Theincrease in leptin levels is compared to that which would otherwiseoccur (i.e., occur in the absence of the FXR agonist administration).

While not wishing to be held to a single theory, the present inventorsbelieve that administration of an FXR agonist results in increasedexpression of genes that play a role in metabolism and weightmaintenance. FXR agonists are shown herein to increase expression ofFGF19 in human cells (hepatocytes). A protein homologous to FGF19, orhaving high sequence similarity to FGF19, may not naturally occur in allmammals. However it is postulated that non-human mammals possessfibroblast growth factors (or biomolecules having similar functionaleffects) that provide an FXR-mediated pathway similar to the FXR-FGF19pathway, such that administration of FXR agonists to the animal willaffect adipocyte function, metabolism and/or overall weight maintenance(or weight loss) in the manner described herein.

Leptin is a hydrophilic protein secreted from white adipocytes.Administration of recombinant leptin to ob/ob mice, which lack thefunctional protein and are obese, results in a reduction of food intakeand an increase in energy expenditure. Halas et al., Science 269:540-549 (1995). Insulin, glucocorticoids, TNFα, and interleukin-1 havebeen reported to stimulate the expression of the ob gene; fasting andthe administration of isoprenaline or selective β3-adrenoceptor agonistshave been reported to cause a decrease in ob gene expression and acorresponding decrease in circulating leptin. Serum concentrations ofleptin have been reported as reflecting the nutritional status and bodyfat mass of individuals.

The present invention further provides a method of decreasing glucoseuptake by the cells of a mammal (particularly adipocytes or whiteadipocyte cells) by administering an FXR agonist to the mammal in anamount effective to decrease glucose uptake by such cells. The decreasein glucose uptake is relative to that which would occur in the absenceof the FXR agonist treatment.

The present invention further provides a method of treating a mammaliansubject in need of weight loss or weight maintenance treatment, byadministering to the subject a pharmaceutically acceptable FXR agonistin an amount effective to decrease said subject's weight. The decreasein weight is relative to the change in weight which would occur (orwould be expected to occur) in the absence of FXR agonist treatment. Thepresent methods include pharmaceutical weight loss treatment of humans.

The present uses of FXR agonists include methods of increasing themetabolic rate of a mammalian subject, or increasing serum (orcirculating) leptin in a mammalian subject, comprising administering toa subject a pharmaceutically acceptable FXR agonist in an amounteffective to increase the metabolic rate or increase the serum leptinconcentrations of the subject. The increase in metabolic rate may beassessed by any suitable means as is known in the art, such as bymeasuring oxygen consumption and/or urine output in a controlledsetting. The increase in metabolic rate or leptin concentration isrelative to that would occur in the absence of FXR agonist treatment.

While not wishing to be held to a single theory underlying the presentinvention, the present inventors believe that, in a mammal in which bothFarnesoid X Receptors and FGF19 (or a protein having similar function toFGF19) are naturally present, administration of an FXR agonist to theanimal will increase levels of the fibroblast growth factor (or similarprotein), will affect adipocytes (including but not limited to increasedrelease of leptin and/or decreased uptake of glucose), and will affectthe animal's overall metabolic and/or weight status.

Also provided herein is a method of inducing FGF19 expression from humancells, preferably human hepatocytes, by administering an FXR agonist tosaid cell or exposing said cell to an FXR agonist; the cells may be invitro. FGF19 protein secreted into cell culture media may be isolatedand purified using any suitable technique as is known in the art. Allpublications and references, including but not limited to patents andpatent applications, cited in this specification are herein incorporatedby reference in their entirety as if each individual publication orreference were specifically and individually indicated to beincorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

EXAMPLES Example 1 Differential Expression of FGF19 in Human HepatocytesTreated with FXR Agonists

The pattern of gene expression induced in human hepatocytes aftertreatment with an FXR ligand was measured.

Materials and Methods

Nine samples of human hepatocytes were used: Human hepatocytes treatedwith DMSO control (3 samples), human hepatocytes treated withchenodeoxycholic acid (CDCA; 3 samples), and human hepatocytes treatedwith FXR agonist GW4064X (3 samples). RNA was extracted from the cellsand gene expression analysis was performed by CuraGen Corporation (NewHaven, Conn.) using transcript profiling technology as described above.See Nat Biotechnol 17:798-803, 1999; see also U.S. Pat. Nos. 5,871,697and 5,972,693.

Three independent reactions from each cDNA sample were compared forquality of electrophoretic peak resolution and reproducibility of peakpatterns. Composite traces from each sample were generated, and thencompared among the three independent samples for peak quality andreproducibility. The resulting traces represent the total geneexpression profile for the tissue sampled and treatment. The databasesfor each sample were compared to identify differences in gene expressionresulting from the different treatments. The composite traces calculatedfor each sample group, based on average peak height and variance, werecompared among sample groups using software designed to identify peaksrepresenting differential expression.

Bands (representing gene fragments) that did not change expressionlevels in either of the treatments (compared to control group) werefiltered out. Bands showing differential expression in at least one ofthe treatments by a factor greater than or equal to +/−1.5 fold(compared to control) were included in the analysis.

Results

Expression of human Fibroblast Growth Factor 19 (hFGF19) was increased36.4-fold in human hepatocytes treated with FXR agonist compound GW4064Xcompared to control cells; expression of hFGF19 was increased 5.6-foldin human hepatocytes treated with CDCA compared to control cells. Theseresults indicated that treatment of a cell with an FXR agonist increasestranscription of hFGF19.

Example 2 Effect of FXR Agonist on Leptin Release from Adipocytes InVitro

The addition of recombinant human FGF-19 to cultures of primary ratadipocytes is reported to increase the release of leptin from the cells(WO 0118210). As noted in Example 1, herein, expression of humanFibroblast Growth Factor 19 (hFGF19) was increased in human hepatocytestreated with FXR agonist compounds, compared to control cells.

The present study investigates the use of an FXR agonist to induceexpression of FGF-19 in liver cells. Increased secretion of the hFGF19protein can thereby increase the release of leptin from adipocytes.

Cultures of rat adipocytes are established using any suitable means asis known in the art. One suitable method harvests fibroblasticpreadipocytes from the inguinal fat deposit of sucking rats, which arethen cultured and induced to differentiate into mature adipocytes.Following differentiation, the ob (leptin) gene is expressed and leptinis secreted into the culture medium (typically by day 4 afterinduction). Mitchell et al., Biochem Biophys Res Comm, 230:360 (1997).Alternatively, isolated primary adipocytes may be cultured (see, e.g.,Hardie et al., Horm Metab Res 28:685 (1996)), or excised pieces ofadipose tissue may be maintained in cell culture conditions (see, e.g.,Ott et al., Exp. Biol. Med. 226(9):841 (2002)).

An FXR agonist is added to liver cells cultures (primary humanhepatocytes or HuH7 cells) in order to induce expression and secretionof FGF19. Suitable FXR agonists include compounds of Formulas I-III asdescribed herein. The resulting conditioned media containing FGF19 isthen added to adipocyte cell cultures in a range of concentrations.Control cell cultures are also prepared (without FXR agonist), toprovide control media which is added to control cultures of adipocytecells.

Leptin secreted into culture medium from the adipocyte cell cultures ismeasured by any suitable means as is known in the art, including e.g.,Enzyme Linked Immunosorbent Assay (ELISA) or Radio-Immunoassay (RIA)(Rat Leptin RIA kit, Linco Research Inc., St. Charles, Mo.). Leptinsecretion is measured as a function of FXR agonist concentration,(and/or conditioned media concentration, and/or FGF19 concentration) andtime elapsed after addition of conditioned media to adipocyte cellcultures, and is compared to control cell cultures.

Example 3 Effect of FXR Agonist on Glucose Uptake by Adipocytes In Vitro

The addition of recombinant human FGF-19 to cultures of primary ratadipocytes has been reported to decrease the uptake of glucose (WO0118210). The present study investigates the use of an FXR agonist toinduce expression of FGF-19 in liver cells, and the effect of hFGF19protein on the uptake of glucose by rat white adipocytes.

Cultures of primary rat adipocytes and human liver cells are establishedusing any suitable means as is known in the art, as discussed above.

An FXR agonist is added to liver cell cultures (primary humanhepatocytes or HuH7 cells) to induce expression and secretion of FGF19.Control liver cell cultures (without exposure to FXR agonist) are alsoprepared. Suitable FXR agonists include compounds of Formulas I-III asdescribed herein. Conditioned media obtained from the liver cellcultures exposed to FXR agonist (and control medium) are then added toadipocyte cell cultures in a range of concentrations. Glucose uptake byadipocytes is measured by any suitable means as is known in the art, asa function of FXR agonist concentration (and/or conditioned mediaconcentration, and/or FGF19 concentration) and time elapsed afteraddition of FXR agonist, and is compared to control cell cultures.

Example 4 FXR Agonist in Mice

Infusion of FvB mice with recombinant human FGF-19 (1 mg/kg/day,delivered intravenously by an osmotically driven implanted pump) hasbeen reported to result in increased food intake and increased oxygenconsumption compared to mice infused with carrier alone (WO 01 18210,Genentech).

Non-transgenic FvB mice are administered an FXR agonist in a suitablecarrier; control mice are administered the carrier alone. Suitable FXRagonists include compounds of Formulas I-III as described herein.Administration may be by any means suitable for the particular FXRagonist (e.g., oral, subcutaneous, intravenous, or via implanted pump).Mice receiving FXR agonist may be further divided into groups receivingvarying dosage regimes of FXR agonist. Treated and control mice may befurther divided according to feeding regime (e.g., ad libitum normaldiet; controlled portion normal diet; ad libitum high fat diet;controlled portion high fat diet).

Weight, and optionally oxygen consumption, of the treated mice and thecontrols are measured at various time points and compared. Weight may bemeasured by any suitable means, including total animal weight and/orsacrifice of the animals after a set amount of time and measurement ofthe weight of specific fat deposits (e.g., epididymal, retroperitonealwith peri-renal). Circulating leptin may also be measured at varioustime points and compared among treatment group(s) and controls. Adecrease in weight, or decreased rate of weight gain, and/or decrease inadiposity (as indicated by circulating leptin levels), in treated micerelative to controls indicates that the FXR agonist decreases adiposity,and indicates FXR agonists as a therapeutic in the treatment of obesityin mammals.

Example 5 FXR Agonist in Rats

A strain of rat suitable for use in laboratory experiments and not knownto have metabolic defects are administered an FXR agonist in a suitablecarrier and at a range of FXR dosages; control rats are administered thecarrier alone. Suitable FXR agonists include compounds of Formulas I-IIIas described herein. Administration may be by any means suitable for theparticular FXR agonist (e.g., oral, subcutaneous, intravenous, or viaimplanted pump). Treated and control rats may be further dividedaccording to feeding regime (e.g., ad libitum normal diet; controlledportion normal diet; ad libitum high fat diet; controlled portion highfat diet).

Weight, and optionally oxygen consumption, of the treated animals andthe controls are measured at various time points and compared. Weightmay be measured by any suitable means, including total animal weightand/or sacrifice of the animals after a set amount of time andmeasurement of the weight of specific fat deposits (e.g., epididymal,retroperitoneal with peri-renal). Circulating leptin may also bemeasured at various time points and compared among treatment group(s)and controls. A decrease in weight, or decreased rate of weight gain,and/or decrease in adiposity (as indicated by circulating leptinlevels), in treated animals relative to controls indicates that the FXRagonist decreases adiposity, and indicates FXR agonists as a therapeuticin the treatment of obesity in mammals.

Example 6 FXR Agonist in Primates

A group of non-human primates suitable for use in laboratory experimentsand not known to have any metabolic defects are administered an FXRagonist in a suitable carrier and at a range of FXR dosages; controlprimates are administered the carrier alone. Suitable FXR agonistsinclude compounds of Formulas I-III as described herein. Administrationmay be by any means suitable for the particular FXR agonist (e.g., oral,subcutaneous, intravenous, or via implanted pump). Treated and controlanimals may be further divided according to feeding regime (e.g., adlibitum normal diet; controlled portion normal diet; ad libitum high fatdiet; controlled portion high fat diet).

Weight, and optionally oxygen consumption, of the treated animals andthe controls are measured at various time points and compared. Weightmay be measured by any suitable means, including total animal weightand/or sacrifice of the animals after a set amount of time andmeasurement of the weight of specific fat deposits (e.g., epididymal,retroperitoneal with peri-renal). Circulating leptin or expression ofFGF19 may also be measured at various time points and comparedamongtreatment group(s) and controls. A decrease in weight, or decreased rateof weight gain, and/or decrease in adiposity (as indicated bycirculating leptin levels), in treated animals relative to controlsindicates that the FXR agonist decreases adiposity, and indicates FXRagonists as a therapeutic in the treatment of obesity in mammals.

1. A method of increasing leptin release from adipocyte cells of amammalian subject, comprising administering an FXR agonist to saidsubject in an amount effective to increase leptin release, compared tothat which would occur in the absence of said FXR agonistadministration.
 2. A method of decreasing glucose uptake by adipocytecells of a mammalian subject, comprising administering an FXR agonist tosaid subject in an amount effective to decrease glucose uptake comparedto that which would occur in the absence of said FXR agonistadministration.
 3. A method of treating a mammalian subject in need ofweight loss treatment, comprising administering to said subject apharmaceutically acceptable FXR agonist in an amount effective todecrease said subject's weight, compared to the weight loss that wouldoccur in the absence of FXR agonist treatment.
 4. A method of reducingtotal body mass of a mammalian subject, comprising administering to asubject a pharmaceutically acceptable FXR agonist in an amount effectiveto reduce said subject's total body mass, compared to the subject'stotal body mass that would occur in the absence of FXR agonisttreatment.
 5. A method of increasing the metabolic rate of a mammaliansubject, comprising administering to a subject a pharmaceuticallyacceptable FXR agonist in an amount effective to increase the metabolicrate of said subject, compared to the rate that would occur in theabsence of FXR agonist treatment.
 6. A method of increasing serum leptinin a mammalian subject, comprising administering to a subject apharmaceutically acceptable FXR agonist in an amount effective toincrease serum leptin in said subject, compared to that would occur inthe absence of FXR agonist treatment.
 7. A method according to claim 1,wherein said FXR agonist is selected from the group consisting ofGW4064; 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid;3α,7α-dihydroxy-6α-propyl-5β-cholan-24-oic acid; and3α,7α-dihydroxy-6α-allyl-5β-cholan-24-oic acid, and pharmaceuticallyacceptable salts, solvates or amino acid conjugates thereof. 8.(Canceled)
 9. (Canceled)
 10. (Canceled)
 11. A method of inducingexpression of FGF19 in a human hepatocyte cell, comprising administeringan FXR agonist to said cell.
 12. A method according to claim 11, whereinsaid FXR agonist is selected from the group consisting of GW4064;3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid;3α,7α-dihydroxy-6α-propyl-5β-cholan-24-oic acid; and3α,7α-dihydroxy-6α-allyl-5β-cholan-24-oic acid, and pharmaceuticallyacceptable salts, solvates or amino acid conjugates thereof.
 13. Amethod according to claim 11 where said cell is in vitro.
 14. A methodaccording to claim 2, wherein said FXR agonist is selected from thegroup consisting of GW4064; 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oicacid; 3α,7α-dihydroxy-6α-propyl-5β-cholan-24-oic acid; and3α,7α-dihydroxy-6α-allyl-5β-cholan-24-oic acid, and pharmaceuticallyacceptable salts, solvates or amino acid conjugates thereof.
 15. Amethod according to claim 3, wherein said FXR agonist is selected fromthe group consisting of GW4064;3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid;3α,7α-dihydroxy-6α-propyl-5β-cholan-24-oic acid; and3α,7α-dihydroxy-6α-allyl-5β-cholan-24-oic acid, and pharmaceuticallyacceptable salts, solvates or amino acid conjugates thereof.
 16. Amethod according to claim 3, wherein said FXR agonist is selected fromthe group consisting of GW4064;3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid;3α,7α-dihydroxy-6α-propyl-5β-cholan-24-oic acid; and3α,7α-dihydroxy-6α-allyl-5β-cholan-24-oic acid, and pharmaceuticallyacceptable salts, solvates or amino acid conjugates thereof.
 17. Amethod according to claim 4, wherein said FXR agonist is selected fromthe group consisting of GW4064;3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid;3α,7α-dihydroxy-6α-propyl-5β-cholan-24-oic acid; and3α,7α-dihydroxy-6α-allyl-5β-cholan-24-oic acid, and pharmaceuticallyacceptable salts, solvates or amino acid conjugates thereof.
 18. Amethod according to claim 5, wherein said FXR agonist is selected fromthe group consisting of GW4064;3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid;3α,7α-dihydroxy-6α-propyl-5β-cholan-24-oic acid; and3α,7α-dihydroxy-6α-allyl-5β-cholan-24-oic acid, and pharmaceuticallyacceptable salts, solvates or amino acid conjugates thereof.
 19. Amethod according to claim 6, wherein said FXR agonist is selected fromthe group consisting of GW4064;3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid;3α,7α-dihydroxy-6α-propyl-5β-cholan-24-oic acid; and3α,7α-dihydroxy-6α-allyl-5β-cholan-24-oic acid, and pharmaceuticallyacceptable salts, solvates or amino acid conjugates thereof.